1. Field of the Invention
The present invention relates to hybridomas producing anti-methylated DNA antibodies, monoclonal antibodies produced by the hybridomas and a method for immunoprecipitation of methylated DNA by using the antibodies.
2. Description of the Related Art
Chromosome DNAs of higher eukaryotes may sometimes undergo methylation at the 5-position of C (cytosine) among other bases constituting DNAs. Such DNA methylation in higher eukaryotes functions as a mechanism for suppression of expression of genetic information. For example, when a region containing many CpGs (also referred to as CpG islands), which is often found in promoter regions of certain genes, is methylated, transcription of these genes may be suppressed. On the other hand, when a CpG island is not methylated, a transcription factor can bind to the promoter region and the gene can be transcribed.
Accordingly, DNA methylation is one of control mechanisms of gene expression. DNA methylation plays important roles in various physiological and pathological phenomena such as early embryonic development, expression of tissue specific genes, genomic imprinting and X chromosome inactivation which are characteristic to mammals, stabilization of chromosomes, synchronization of DNA replication and the like.
Recently, it has also been revealed that DNA methylation is deeply involved in cancers or other diseases. Thus, studies have been carried out on confirmed diagnosis or prognostic prediction of cancer based on methylation analysis of various genes.
One of the known techniques for recovery of methylated DNA is Methylated DNA Immunoprecipitaion (MeDIP method). According to the MeDIP method, methylated DNA is immunoprecipitated with an antibody specifically recognizing methylated DNA or with a methylated DNA-binding protein, thereby concentrating methylated DNA in a sample. It has also been known that the MeDIP method can be combined with microarray analysis and the thus combined MeDIP-chip method can be used for exhaustive analysis of DNA methylation status.
Various anti-methylated DNA antibodies to be used in the MeDIP method have been commercially available, among which antibodies recognizing 5-methyl cytosine or 5-methyl cytidine have been frequently used, for example. Japanese Unexamined Patent Application Publication No. 2003-125766 discloses that 5-methyl-2′-deoxycytidine is used as an antigen to generate hybridomas from which a monoclonal antibody is obtained and the antibody may be used in enzyme-linked immunosorbent assay (ELISA) to quantify 5-methyl-2′-deoxycytidine.